Rat pheochromocytoma PC12 cells and bovine adrenal chromaffin cells are used to study the mechanism of secretion and its regulation by calcium and GTP-binding proteins. Our goals are to determine how calcium induces secretion and to identify proteins involved in the final steps of the secretory process. Secretion of norepinephrine by permeabilized PC12 cells can be stimulated by the addition of either calcium or GTPgammaS. Stimulation of secretion by GTPgammaS results from its binding to and activating a low molecular weight GTP-binding protein. This GTP- binding protein appears to interact with nucleoside diphosphate kinase. We have used immunoprecipitation, affinity chromatography, and add back experiments to try to isolate this GTP-binding protein. Nucleoside diphosphate kinase appears to be phosphorylated under conditions that stimulate exocytosis in permeabilized PC12 cells. To understand the nature of this phosphorylation, we have examined both the autophosphorylation of nucleoside diphosphate kinase and its ability to function as a protein kinase. As has been reported by others, we found that nucleoside diphosphate kinase autophosphorylates on a histidine residue at its catalytic site. Autophosphorylated nucleoside diphosphate kinase and a cytosolic extract of PC12 cells were used to examine the possibility that this enzyme is a protein kinase. Nucleoside diphosphate kinase directly transferred a phosphate from its catalytic histidine to a histidine on another protein. This protein was purified and shown to be ATP-citrate lyase. ATP-citrate lyase is the primary source of cytosolic acetyl-CoA which is used in a variety of biosynthetic pathways including lipogenesis and cholesterogenesis. Preliminary experiments suggest that nucleoside diphosphate kinase can phosphorylate several other proteins.